Hemophilia A is an X-linked bleeding disorder caused by mutations and/or deletions in the Factor VIII (FVIII) gene resulting in a deficiency of FVIII activity (Peyvandi, F. et al. Haemophilia 12:82-89 (2006). The disease is characterized by spontaneous hemorrhage and excessive bleeding after trauma. Over time, the repeated bleeding into muscles and joints, which often begins in early childhood, results in hemophilic arthropathy and irreversible joint damage. This damage is progressive and can lead to severely limited mobility of joints, muscle atrophy and chronic pain (Rodriguez-Merchan, E. C., Semin. Thromb. Hemost. 29:87-96 (2003), which is herein incorporated by reference in its entirety).
The A2 domain is necessary for the procoagulant activity of the FVIII molecule. Studies show that porcine FVIII has six-fold greater procoagulant activity than human FVIII (Lollar & Parker, J. Biol. Chem. 266:12481-12486 (1991)), and that the difference in coagulant activity between human and porcine FVIII appears to be based on a difference in amino acid sequence between one or more residues in the human and porcine A2 domains (Lollar, P., et al., J. Biol. Chem. 267:23652-23657 (1992)), incorporated herein by reference in its entirety.
Treatment of hemophilia A is by replacement therapy targeting restoration of FVIII activity to 1 to 5% of normal levels to prevent spontaneous bleeding (Mannucci, P. M., et al., N. Engl. J. Med. 344:1773-1779 (2001), which is herein incorporated by reference in its entirety). e.g.
Plasma-derived FVIII (pdFVIII) and recombinant human FVIII (rFVIII) products are utilized for treatment (on-demand therapy) and prevention (prophylaxis therapy) of bleeding episodes. rFVIII was developed to reduce the risk of blood-borne pathogen transmission following the widespread contamination of plasma products with HIV and hepatitis viruses, and to secure an adequate supply of FVIII product. However, hemostatic protection with current FVIII products is temporally limited due to a short half-life (t1/2) of approximately 8-12 hours, requiring prophylactic injections three times per week or every other day for most patients in order to maintain FVIII levels above 1%, a level that has been established as protective against most spontaneous bleeding episodes. Manco-Johnson et al., New Engl J Med. 357(6):535-44 (2007).
Many studies have shown that, even at high doses, on-demand therapy is not effective in preventing arthropathy. Aledort L. et al., J Intern Med. 236:391-399 (1994); Petrini P. et al., Am J Pediatr Hematol Oncol. 13:280-287 (1991). The benefits of prophylactic therapy have been demonstrated in numerous clinical studies. Aznar J. et al., Haemophilia 6(3):170-176 (2000), Feldman B. et al., J Thromb Haemost. 4:1228-1236 (2006), Kreuz W. et al., Haemophilia 4:413-417 (1998), Liesner R. et al., B J Haem. 92:973-978 (1996), Ljung R., Haemophilia. 4(4):409-412 (1998), Löfquist T, et al., J Intern Med 241:395-400 (1997), Nilsson I, et al., B. J Int Med 232:25-32 (1992), Risebrough N. et al., Haemophilia. 14:743-752 (2008), Van Den Berg H. et al., Haemophilia 9 (Suppl. 1):27-31 (2003), Van Den Berg H. et al., Haematologica 89(6):645-650 (2004) and Manco-Johnson et al., supra, established that children started on primary prophylaxis after their first joint bleed had significantly fewer bleeds and less joint damage than children treated on-demand.
Compared to on-demand treatment, prophylactic therapy also decreases disability, hospitalization rate, and time lost from school or work; Aznar J. et al., Haemophilia 6(3):170-176 (2000), Molho P. et al., Haemophilia 6(1):23-32 (2000) and improves quality of life for patients and their families. Coppola A. et al., Blood Transfus. 6(2): 4-11 (2008). However, prophylactic therapy often requires use of central venous access devices in children, and their attendant risks of infection, sepsis, and thrombosis. In addition, despite the benefits, acceptance of and compliance with prophylaxis decreases with age, in part because of inconvenience and invasiveness. Geraghty S. et al., Haemophilia 12:75-81 (2006), Hacker M. et al., Haemophilia 7(4):392-396 (2001). Thus, an rFVIII product with a prolonged plasma t1/2 would potentially be of benefit. Lillicrap D., Current Opinion in Hematology 17:393-397 (2010).
Reduced mortality, prevention of joint damage, and improved quality of life have been important achievements due to the development of pdFVIII and rFVIII. Prolonged protection from bleeding would represent another key advancement in the treatment of hemophilia A patients. However, to date, no products that allow for prolonged hemostatic protection have been developed. Therefore, there remains a need for improved methods of treating hemophilia due to FVIII deficiency that are more tolerable, longer lasting, and more effective than current therapies.
In addition, 15-30% of previously untreated patients develop neutralizing anti-FVIII antibodies (inhibitors) after transfusion with FVIII products. Various techniques for avoiding such immune responses have been considered. These techniques include high-dose tolerance protocols, use of peptide decoys mimicking the anti-FVIII antibody, bypassing immune recognition with human/porcine FVIII hybrid molecules, neutralizing FVIII-reactive CD4 T-cells with anticlonotypic antibodies, using universal CD4 epitopes, and blocking costimulation of CTLA-4-Ig or anti-CD40L. See, e.g., Lei et al., Transfusion Medicine 105: 4865-4870 (2005). Presentation of FVIII by immune cells in order to induce tolerance has also been studied. For example, Lei et al. found that presentation of FVIII domains on an Ig backbone in B cells prevented or decreased antibodies. Id. In addition, Qadura et al. found that tolerogenic presentation of FVIII using immature dendritic cells may reduce immunogenicity. Journal of Thrombosis and Haemostatis 6: 2095-2104 (2008). However, such methods are costly, complicated (e.g., by requiring co-administration of other therapeutics in combination with FVIII or administration of whole cells instead of relatively simple proteins), likely to result in unwanted side-effects, and/or inefficient. Accordingly, there remains a need for simple methods of treating hemophilia due to FVIII deficiency that are effective in patients that develop inhibitory responses.